DESCRIPTION: The overall goal of the proposed research is to reveal how ribonucleases promote tumor cell death. Onconase is the first cancer therapeutic based on the cytotoxicity of ribonucleases. Onconase is a homologue of the well-characterized enzyme ribonuclease A, which is not cytotoxic. The three dimensional structures of these two enzymes diverge in two significant ways - the site in RNase A that binds to the ribonuclease inhibitor protein RI, endogenous to mammalian cells, differs from the analogous region in onconase, and RNase A has an anionic patch absent in onconase. The working hypothesis of this proposal is that these two differences are necessary for the cytotoxic activity of onconase. This hypothesis will be tested by using RNase A as a template for the construction of hybrid proteins. Because RNase A lacks cytotoxic activity any such activity in a hybrid protein would reveal a biochemical property necessary for cytotoxicity. The specific aims of the proposed research are to determine: (1) the role of RI binding by disrupting the RI binding site of RNase A; (2) role of surface charge by eliminating the anionic patch on RNase A; (3) intracellular routing by attaching a KDEL tail or fluorescent probe to RNase A to discern the path to cellular RNA; and (4) intracellular RI levels by using immobilized RNase A to assess the level of metabolically radiolabeled RI in different cell types. Relevant properties of each mutant RNase A will be assessed in comparison to onconase and wild-type RNase A. These properties include cytotoxicity, ribonucleolytic activity, affinity for RI, pI and thermal stability. Appropriate mutations will be combined with the goal of enhancing cytotoxicity. Finally, the three-dimensional structures of mutant RNase As with notable cytotoxicity will be determined by X-ray diffraction analysis. The proposed research is designed to reveal new insights into the basis of ribonuclease cytotoxicity and could lead to new ribonucleases for cancer therapy.